Use of tight junction antagonists to treat inflammatory bowl disease

ABSTRACT

The present invention provides materials and methods for the treatment of inflammatory bowel disease (e.g., Crohn&#39;s disease and ulcerative colitis). Materials of the invention may include compositions comprising one or more tight junction antagonists and optionally one or more therapeutic agents. Methods of the invention may comprise treating a subject in need thereof with a composition comprising one or more tight junction antagonists and, optionally one or more therapeutic agents.

This Application is a continuation of application Ser. No. 11/869,230,filed Oct. 9, 2007 now abandoned, which claims the benefit of U.S.Provisional Application No. 60/849,802, filed Oct. 6, 2006, U.S.Provisional Application No. 60/851,318, filed Oct. 13, 2006, and U.S.Provisional Application No. 60/916,412, filed May 7, 2007, each of whichis hereby incorporated by reference in its entirety.

In normal bowels, the immune reaction is regulated to maintainhomeostasis of the gut. Inflammatory bowel disease (IBD) is a phraseused to describe an inappropriate immune response that occurs in thebowels of affected individuals. Two major types of IBD have beendescribed: Crohn's disease (CD) and ulcerative colitis (UC). Both formsof IBD show abnormal profiles of T cell mediated immunity. In the gut ofCD, a strong Th1 reaction is induced, while the Th2 response isupregulated in the colon of UC.

A variety of inflammatory cytokines have been implicated in IBD. Forexample, in UC increased proinflammatory cytokine production isobserved. IL-13 was identified as an important effector cytokine in UCthat impairs epithelial barrier function by affecting epithelialapoptosis, tight junctions, and restitution velocity (Heller, et al.,Gastroenterology 129(2): 550-64, 2005). TNF-α has been implicated in thepathology of CD and antibodies directed against TNF-α have been used totreat CD (see Nakamura, et al. World J Gastroenterol 2006 Aug. 7;12(29): 4628-4635).

The barrier function of the intestines is impaired in IBD. For example,Crohn's disease is associated with increased permeability of theintestinal barrier even in quiescent patients (Oshitani, et al., Int JMol Med 15(3):407-10, 2005). A TNF-α-induced increase in intestinalepithelial tight junction (TJ) permeability has been proposed to be animportant proinflammatory mechanism contributing to intestinalinflammation in Crohn's disease and other inflammatory conditions (seeYe et al., American Journal of Physiology-Gastrointestinal and LiverPhysiology, 290(3):496-504, 2006). Increased intestinal permeabilityduring episodes of active disease correlates with destruction orrearrangement of the tight junction protein complex (Willemsen, et al.Clin. Exp. Immunol. 142(2): 275-284, 2005).

Zonula occludens toxin (ZOT), which is produced by Vibrio cholerae, hasbeen characterized by Fasano et al., (Proc. Natl. Acad. Sci., USA,8:5242-5246 (1991)) and the sequence has been determined (GenBankaccession no. A43864). ZOT increases the intestinal permeability ofrabbit ileal mucosa by modulating the structure of intercellular tightjunctions.

Peptide antagonists of tight junction opening were described in U.S.Pat. No. 6,458,925, which is incorporated by reference herein in itsentirety, which corresponds to WO 00/07609. Peptide antagonists of tightjunction opening may bind to the receptor utilized by the zonnulaoccludens toxin expressed by Vibrio cholerae, yet not function tophysiologically modulate the opening of mammalian tight junctions. Thepeptide antagonists competitively inhibit the binding of ZOT and zonulinto the ZOT receptor, thereby inhibiting the ability of ZOT and zonulinto physiologically modulate the opening of mammalian tight junctions.

The main treatments available for IBD are steroids and immunosuppressiveagents which non-specifically reduce immunity and inflammation. Thesetherapies are prone to undesired side effects. There remains a need inthe art for treatments of IBD. This need and others are met by thepresent invention.

SUMMARY OF THE INVENTION

The present invention provides methods and materials for treatinginflammatory bowel disease. In some embodiments, the invention providesmethods of treating inflammatory bowel disease comprising administeringto a subject in need thereof a composition comprising a tight junctionantagonist. As used herein, a “subject” may be any mammal, for example,a human, dog, cat, horse, cow, etc. In some embodiments, a subject maybe a human. In other embodiments, a subject may be a dog. Any tightjunction antagonist may be used, for example, a tight junctionantagonist of the invention may be a peptide. When a tight junctionantagonist for use in the invention is a peptide, the peptide maycomprise one or more of SEQ ID NOs: 1-24, which may be on the same ordifferent molecules. In some embodiments, a peptide tight junctionantagonist may comprise the sequence GGVLVQPG (SEQ ID NO:15). In someembodiments, the peptide tight junction antagonist may consistessentially of the sequence GGVLVQPG (SEQ ID NO:15). Compositionssuitable for use in treating IBD may be formulated in any manner knownto those skilled in the art. In some embodiments, a composition suitablefor treating IBD may comprise a tight junction antagonist and may be adelayed release composition. Compositions for use in treating IBD,delayed release or otherwise, may comprise one or more tight junctionantagonists and one or more therapeutic agents. Suitable therapeuticagents include, but are not limited to, aminosalicylates,corticosteroids, immunomodulators, antibiotics, and biologictherapeutics. In some embodiments, a composition suitable for treatingIBD may comprise a peptide tight junction antagonist (e.g., a peptidecomprising SEQ ID NO: 15) and a therapeutic agent, e.g., a steroid.

The present invention provides methods and materials for treatingCrohn's disease. In some embodiments, the invention provides methods oftreating Crohn's disease comprising administering to a subject in needthereof a composition comprising a tight junction antagonist. Any tightjunction antagonist may be used, for example, a tight junctionantagonist of the invention may be a peptide. When a tight junctionantagonist for use in the invention is a peptide, the peptide maycomprise one or more of SEQ ID NOs: 1-24, which may be on the same ordifferent molecules. In some embodiments, a peptide tight junctionantagonist may comprise the sequence GGVLVQPG (SEQ ID NO:15). In someembodiments, the peptide tight junction antagonist may consistessentially of the sequence GGVLVQPG (SEQ ID NO:15). Compositionssuitable for use in treating Crohn's disease may be formulated in anymanner known to those skilled in the art. In some embodiments, acomposition suitable for treating Crohn's disease may comprise a tightjunction antagonist and may be a delayed release composition.Compositions for use in treating Crohn's disease, delayed release orotherwise, may comprise one or more tight junction antagonists and oneor more therapeutic agents. Suitable therapeutic agents include, but arenot limited to, aminosalicylates, corticosteroids, immunomodulators,antibiotics, and biologic therapeutics. In some embodiments, acomposition suitable for treating Crohn's disease may comprise a peptidetight junction antagonist (e.g., a peptide comprising SEQ ID NO: 15) anda therapeutic agent, e.g., a steroid.

The present invention provides methods and materials for treatingulcerative colitis. In some embodiments, the invention provides methodsof treating ulcerative colitis comprising administering to a subject inneed thereof a composition comprising a tight junction antagonist. Anytight junction antagonist may be used, for example, a tight junctionantagonist of the invention may be a peptide. When a tight junctionantagonist for use in the invention is a peptide, the peptide maycomprise one or more of SEQ ID NOs: 1-24, which may be on the same ordifferent molecules. In some embodiments, a peptide tight junctionantagonist may comprise the sequence GGVLVQPG (SEQ ID NO:15). In someembodiments, the peptide tight junction antagonist may consistessentially of the sequence GGVLVQPG (SEQ ID NO:15). Compositionssuitable for use in treating ulcerative colitis may be formulated in anymanner known to those skilled in the art. In some embodiments, acomposition suitable for treating ulcerative colitis may comprise atight junction antagonist and may be a delayed release composition.Compositions for use in treating ulcerative colitis, delayed release orotherwise, may comprise one or more tight junction antagonists and oneor more therapeutic agents. Suitable therapeutic agents include, but arenot limited to, aminosalicylates, corticosteroids, immunomodulators,antibiotics, and biologic therapeutics. In some embodiments, acomposition suitable for treating ulcerative colitis may comprise apeptide tight junction antagonist (e.g., a peptide comprising SEQ ID NO:15) and a therapeutic agent, e.g., a steroid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Body Weight area under the curve from 4-17 weeks of age.

FIG. 2 shows a measurement of Gastric Permeability measured as sucroseexcretion.

FIG. 3 shows Small Intestinal Permeability measured by lactulosemannitol test.

FIG. 4 shows Small Intestinal Permeability measured by lactulosemannitol test.

FIG. 5 shows in vitro measurement of colonic permeability at 8 weeks ofage.

FIG. 6 shows in vitro colonic electrical resistance at 8 weeks of age.

FIG. 7 shows colonic inflammation (neutrophil infiltration) at 17 weeks.

FIG. 8 shows colonic inflammation at 17 weeks (TNF secretion over 24hours).

FIG. 9 shows colonic inflammation at 17 weeks (IFN secretion over 24hours).

FIG. 10 shows Colonic Permeability as measured by sucralose excretion.

DETAILED DESCRIPTION OF THE INVENTION Antagonists of Tight JunctionOpening

Any antagonist of tight junction opening may be used in the practice ofthe present invention. As used herein, tight junction antagonistsprevent, inhibit or reduce the opening of tight junctions. A tightjunction antagonist may bind to the zonulin receptor and prevent,inhibit, reduce or reverse the tight junction opening triggered byzonulin. For example, antagonists of the invention may comprise peptideantagonists. Examples of peptide antagonists include, but are notlimited to, peptides that comprise an amino acid sequence selected fromthe group consisting of

Gly Arg Val Cys Val Gln Pro Gly, (SEQ ID NO: 1)Gly Arg Val Cys Val Gln Asp Gly, (SEQ ID NO: 2)Gly Arg Val Leu Val Gln Pro Gly, (SEQ ID NO: 3)Gly Arg Val Leu Val Gln Asp Gly, (SEQ ID NO: 4)Gly Arg Leu Cys Val Gln Pro Gly, (SEQ ID NO: 5)Gly Arg Leu Cys Val Gln Asp Gly, (SEQ ID NO: 6)Gly Arg Leu Leu Val Gln Pro Gly, (SEQ ID NO: 7)Gly Arg Leu Leu Val Gln Asp Gly, (SEQ ID NO: 8)Gly Arg Gly Cys Val Gln Pro Gly, (SEQ ID NO: 9)Gly Arg Gly Cys Val Gln Asp Gly, (SEQ ID NO: 10)Gly Arg Gly Leu Val Gln Pro Gly, (SEQ ID NO: 11)Gly Arg Gly Leu Val Gln Asp Gly, (SEQ ID NO: 12)Gly Gly Val Cys Val Gln Pro Gly, (SEQ ID NO: 13)Gly Gly Val Cys Val Gln Asp Gly, (SEQ ID NO: 14)Gly Gly Val Leu Val Gln Pro Gly, (SEQ ID NO: 15)Gly Gly Val Leu Val Gln Asp Gly, (SEQ ID NO: 16)Gly Gly Leu Cys Val Gln Pro Gly, (SEQ ID NO: 17)Gly Gly Leu Cys Val Gln Asp Gly, (SEQ ID NO: 18)Gly Gly Leu Leu Val Gln Pro Gly, (SEQ ID NO: 19)Gly Gly Leu Leu Val Gln Asp Gly, (SEQ ID NO: 20)Gly Gly Gly Cys Val Gln Pro Gly, (SEQ ID NO: 21)Gly Gly Gly Cys Val Gln Asp Gly, (SEQ ID NO: 22)Gly Gly Gly Leu Val Gln Pro Gly, (SEQ ID NO: 23) andGly Gly Gly Leu Val Gln Asp Gly (SEQ ID NO: 24)

When the antagonist is a peptide, any length of peptide may be used.Generally, the size of the peptide antagonist will range from about 6 toabout 100, from about 6 to about 90, from about 6 to about 80, fromabout 6 to about 70, from about 6 to about 60, from about 6 to about 50,from about 6 to about 40, from about 6 to about 30, from about 6 toabout 25, from about 6 to about 20, from about 6 to about 15, from about6 to about 14, from about 6 to about 13, from about 6 to about 12, fromabout 6 to about 11, from about 6 to about 10, from about 6 to about 9,or from about 6 to about 8 amino acids in length. Peptide antagonists ofthe invention may be from about 8 to about 100, from about 8 to about90, from about 8 to about 80, from about 8 to about 70, from about 8 toabout 60, from about 8 to about 50, from about 8 to about 40, from about8 to about 30, from about 8 to about 25, from about 8 to about 20, fromabout 8 to about 15, from about 8 to about 14, from about 8 to about 13,from about 8 to about 12, from about 8 to about 11, or from about 8 toabout 10 amino acids in length. Peptide antagonists of the invention maybe from about 10 to about 100, from about 10 to about 90, from about 10to about 80, from about 10 to about 70, from about 10 to about 60, fromabout 10 to about 50, from about 10 to about 40, from about 10 to about30, from about 10 to about 25, from about 10 to about 20, from about 10to about 15, from about 10 to about 14, from about 10 to about 13, orfrom about 10 to about 12 amino acids in length. Peptide antagonists ofthe invention may be from about 12 to about 100, from about 12 to about90, from about 12 to about 80, from about 12 to about 70, from about 12to about 60, from about 12 to about 50, from about 12 to about 40, fromabout 12 to about 30, from about 12 to about 25, from about 12 to about20, from about 12 to about 15, or from about 12 to about 14 amino acidsin length. Peptide antagonists of the invention may be from about 15 toabout 100, from about 15 to about 90, from about 15 to about 80, fromabout 15 to about 70, from about 15 to about 60, from about 15 to about50, from about 15 to about 40, from about 15 to about 30, from about 15to about 25, from about 15 to about 20, from about 19 to about 15, fromabout 15 to about 18, or from about 17 to about 15 amino acids inlength.

The peptide antagonists can be chemically synthesized and purified usingwell-known techniques, such as described in High Performance LiquidChromatography of Peptides and Proteins: Separation Analysis andConformation, Eds. Mant et al., C.R.C. Press (1991), and a peptidesynthesizer, such as Symphony (Protein Technologies, Inc); or by usingrecombinant DNA techniques, i.e., where the nucleotide sequence encodingthe peptide is inserted in an appropriate expression vector, e.g., an E.coli or yeast expression vector, expressed in the respective host cell,and purified therefrom using well-known techniques.

Formulations

The compositions of the invention may be formulated for entericdelivery, for example, may comprise one or more coatings, for example,delayed release coating containing one or more enteric agents. A delayedrelease coating is typically substantially stable in gastric fluid andsubstantially unstable (e.g., dissolves rapidly or is physicallyunstable) in intestinal fluid, thus providing for substantial release ofthe tight junction antagonist from the composition in the duodenum orthe jejunum. Typically, compositions comprising a tight junctionantagonist (e.g., peptide antagonist) comprise a pharmaceuticallyeffective amount of the antagonist. The pharmaceutically effectiveamount of antagonist (e.g., peptide antagonist) employed may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual. Dosage regimens may be adjusted to provide the optimumtherapeutic response. For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation.

Compositions of the invention may comprise one or more tight junctionantagonists at a level of from about 0.1 wt % to about 20 wt %, fromabout 0.1 wt % to about 18 wt %, from about 0.1 wt % to about 16 wt %,from about 0.1 wt % to about 14 wt %, from about 0.1 wt % to about 12 wt%, from about 0.1 wt % to about 10 wt %, from about 0.1 wt % to about 8wt %, from about 0.1 wt % to about 6 wt %, from about 0.1 wt % to about4 wt %, from about 0.1 wt % to about 2 wt %, from about 0.1 wt % toabout 1 wt %, from about 0.1 wt % to about 0.9 wt %, from about 0.1 wt %to about 0.8 wt %, from about 0.1 wt % to about 0.7 wt %, from about 0.1wt % to about 0.6 wt %, from about 0.1 wt % to about 0.5 wt %, fromabout 0.1 wt % to about 0.4 wt %, from about 0.1 wt % to about 0.3 wt %,or from about 0.1 wt % to about 0.2 wt % of the total weight of thecomposition. Compositions of the invention may comprise one or moretight junction antagonists at a level of about 0.1 wt %, about 0.2 wt %,about 0.3 wt %, about 0.4 wt %, about 0.5 wt %, about 0.6 wt %, about0.7 wt %, about 0.8 wt %, or about 0.9 wt % based on the total weight ofthe composition.

Compositions of the invention may comprise one or more tight junctionantagonists at a level of from about 1 wt % to about 20 wt %, from about1 wt % to about 18 wt %, from about 1 wt % to about 16 wt %, from about1 wt % to about 14 wt %, from about 1 wt % to about 12 wt %, from about1 wt % to about 10 wt %, from about 1 wt % to about 9 wt %, from about 1wt % to about 8 wt %, from about 1 wt % to about 7 wt %, from about 1 wt% to about 6 wt %, from about 1 wt % to about 5 wt %, from about 1 wt %to about 4 wt %, from about 1 wt % to about 3 wt %, or from about 1 wt %to about 2 wt % of the total weight of the composition. Compositions ofthe invention may comprise one or more tight junction effectors at alevel of about 1 wt %, about 2 wt %, about 3 wt %, about 4 wt %, about 5wt %, about 6 wt %, about 7 wt %, about 8 wt %, or about 9 wt % based onthe total weight of the composition.

The terms “stable in gastric fluid” or “stable in acidic environments”refers to a composition that releases 30% or less by weight of the totaltight junction antagonist in the composition in gastric fluid with a pHof 5 or less, or simulated gastric fluid with a pH of 5 or less, inapproximately sixty minutes. Compositions of the of the invention mayrelease from about 0% to about 30%, from about 0% to about 25%, fromabout 0% to about 20%, from about 0% to about 15%, from about 0% toabout 10%, 5% to about 30%, from about 5% to about 25%, from about 5% toabout 20%, from about 5% to about 15%, from about 5% to about 10% byweight of the total tight junction antagonist in the composition ingastric fluid with a pH of 5, or less or simulated gastric fluid with apH of or less, in approximately sixty minutes. As use herein, “about”used to modify a numerical value means within 10% of the value.Compositions of the invention may release about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%by weight of the total tight junction antagonist in the composition ingastric fluid with a pH of 5 or less, or simulated gastric fluid with apH of 5 or less, in approximately sixty minutes.

The term “unstable in intestinal fluid” refers to a composition thatreleases 70% or more by weight of the total tight junction antagonist inthe composition in intestinal fluid or simulated intestinal fluid inapproximately sixty minutes. The term “unstable in near neutral toalkaline environments” refers to a composition that releases 70% or moreby weight of the total amount of tight junction antagonist in thecomposition in intestinal fluid with a pH of 5 or greater, or simulatedintestinal fluid with a pH of 5 or greater, in approximately ninetyminutes. For example, a composition that is unstable in near neutral oralkaline environments may release 70% or more by weight of a tightjunction antagonist peptide in a fluid having a pH greater than about 5(e.g., a fluid having a pH of from about 5 to about 14, from about 6 toabout 14, from about 7 to about 14, from about 8 to about 14, from about9 to about 14, from about 10 to about 14, or from about 11 to about 14)in from about 5 minutes to about 90 minutes, or from about 10 minutes toabout 90 minutes, or from about 15 minutes to about 90 minutes, or fromabout 20 minutes to about 90 minutes, or from about 25 minutes to about90 minutes, or from about 30 minutes to about 90 minutes, or from about5 minutes to about 60 minutes, or from about 10 minutes to about 60minutes, or from about 15 minutes to about 60 minutes, or from about 20minutes to about 60 minutes, or from about 25 minutes to about 90minutes, or from about 30 minutes to about 60 minutes.

In addition to a tight junction antagonist, compositions of theinvention may further comprise one or more therapeutic agents.Therapeutic agents include, but are not limited to, steroids and otheranti-inflammatory compounds. Suitable therapeutic agents may include oneor more of aminosalicylates, corticosteroids, immunomodulators,antibiotics, and biologic therapies. Examples of suitable therapeuticagents that may be included in the compositions of the invention totreat IBD (e.g., Crohn's disease and/or ulcerative colitis) include, butare not limited to:

5-amino salicyclic acid (5-ASA) agents (e.g., Sulfasalazine),Azulfidine®, Asacol®, Dipentum®, Pentasa®, and Rowasa®;

Antibiotics, for example, metronidazole (Flagyl®) and ciprofloxacin(Cipro®), although there are many others that may be effective incertain individuals;

Steroids, e.g., corticosteroids. Suitable steroids include, but are notlimited to, prednisone, hydrocortisone, Medrol®, and budesonidemultiple-release capsule MRC (EntocortREC®)

6-mercaptopurine (6-MP, Purinethol®) and azathioprine (Imuran®); andantibodies against inflammatory cytokines, e.g., Infliximab (Remicade®).

Compositions of the invention may also comprise one or morepharmaceutically acceptable excipients. Suitable excipients include, butare not limited to, buffers, buffer salts, bulking agents, salts,surface active agents, acids, bases, and binders.

Methods of Use

The compositions of the invention can be used for preventing, slowingthe onset of, ameliorating and/or treating IBD (e.g., Crohn's diseaseand/or ulcerative colitis). In one embodiment, the present inventionprovides a method of treating Crohn's disease by administering acomposition comprising one or more tight junction antagonists. In oneembodiment, the present invention provides a method of treatingulcerative colitis by administering a composition comprising one or moretight junction antagonists.

In some embodiments, compositions of the invention may be givenrepeatedly over a protracted period, i.e., may be chronicallyadministered. Typically, compositions may be administered one or moretimes each day in an amount suitable to prevent or reduce the likelihoodof an attack of IBD (e.g., Crohn's disease and/or ulcerative colitis).Such compositions may be administered chronically, for example, one ormore times daily over a plurality of days.

In some embodiments, compositions of the invention may be use to treatacute IBD (e.g., Crohn's disease and/or ulcerative colitis) attacks.Typically, embodiments of this type will require administration of thecompositions of the invention to a subject undergoing an attack in anamount suitable to reduce the severity of the attack. One or moreadministration may be used.

EXAMPLES

Effect of AT-1001 on intestinal permeability and colitis in the IL-10 KOmouse (129/Sve/IL-10KO)

The purpose of this study is to determine the ability of AT-1001,administered daily in drinking water to alter intestinal permeability(measured by absorption/excretion of lactulose and mannitol) and toinhibit the development of colitis in the 129/Svev/IL-10 KO mouse.

Anticipated* Suc/lac/Man/ # Content Dose, Sucralose Animals AnimalAT-1001 (weight/ AT-1001 Dose (0.2 ml sugar per Group ID's TreatmentFormulation vehicle) mg/day probe gavage) Group 1 129/Svev/ Control — —0 Sucrose 500 mg/ml 12 IL-10KO Lactulose 60 mg/ml 1M001-1M012 Mannitol40 mg/ml Sucralose 30 mg/ml 2 129/Svev/ Gavage of — — 0 Sucrose 500mg/ml 12 IL-10KO probiotic Lactulose 60 mg/ml 2M001-2M012 conditionedMannitol 40 mg/ml media Sucralose 30 mg/ml 3 129/Svev/ Low dose of NeatAT-1001 0.2 Sucrose 500 mg/ml 12 IL-10KO AT-1001 Chemical in 0.1 mg/mlLactulose 60 mg/ml 3M001-3M012 autoclaved, H₂O Mannitol 40 mg/ml H₂OSucralose 30 mg/ml 4 129/Svev/ High dose of Neat AT-1001 2 Sucrose 500mg/ml 12 IL-10KO AT-1001 Chemical in 1 mg/ml Lactulose 60 mg/ml4M001-4M012 autoclaved, H₂O Mannitol 40 mg/ml H₂O Sucralose 30 mg/ml 5129/Svev/ Control for — — 0 — 4 IL-10KO baseline zonulin levelmeasurement *assumes 2 ml/day water consumption

Dose Administration

Method and AT-1001 neat chemical in drinking water, and RoutesSuc/lac/man/sucralose given by gavage Dosing Suc/lac/man/suralose:(Groups 1-4) Timepoints 1. Weeks 1-2 Suc/lac/man/suralose solution ondays 3, 6, and 9 2. Weeks 3-15 Suc/lac/man/suralose solution first dayof every week Probiotic conditioned media: (Group 2) Daily in themornings (Start Day 1 of study) AT-1001 Neat Chemical 0.1 mg/ml: (Group3) Ad libidum (Start Day 1 of study) AT-1001 Neat Chemical 1 mg/ml:(Group 4) Ad libidum (Start Day 1 of study) Duration Eighty daysFrequency AT-1001: continuously. Suc/lac/man/sucralose: Days 3, 6, 9,14, 21, 28, 35, 41, 49, 56, 63, 70, 77, 84 Volume Suc/lac/man/sucralose0.2 mL gavage per animal

Dose Administration Details

AT-1001 neat chemical will be administered ad libidum every day startingat day 1 to Group 3 at 0.1 mg/ml and Group 4 at 1 mg/ml in sterile watervia the drinking water supply. Dosing of AT-1001 will be continued whenthe animals are in the metabolic cages for 22 hours. Probioticconditioned medium will be given every morning to Group 2. The solutionis prepared by dissolving 0.01 g in 10 ml of MRS medium and incubatingit at 37° C. for 24 h. After incubation, the tube will be centrifuged 10minutes at 10,000 rpm. The supernatant will be filtered through a 0.22micron filter and diluted 1:10 with MRS medium. Animals will receive 30μl of this dilution every morning. The conditioned media must beprepared fresh every morning. Sucrose/lactulose/mannitol/sucralosesolution is prepared by dissolving Sucrose (50 mg), lactulose (6 mg),mannitol (4 mg), and sucralose (3 mg) in water (100 mL).

Each week, food and water will be removed 4 hours prior to gavage. Allthe collection vials will have 100 μl of paraffin oil to avoid urineevaporation and 100 μl of thymol (10% m/w in propanol). Each animal willbe gavaged with 0.2 ml of sugar solution, and placed in metabolic cageswith access to H₂O only for 22 hours. For the duration of the 22 hours,urine will be collected into previously weighed vials. At the end of thecollection, the funnel of the cages will be washed off with 2 ml ofwater to collect any sugar that may have dried out before reaching thecollection tube. After the collection of urine is complete, the animalswill be placed in their respective cages, and provided with food andwater. Each tube will be weighed to determine volume of urine collected.For the first 2 weeks, the animals will be handled, administeredSuc/lac/man/suralose solution, and introduced to the cages 3 timesduring 10 days. After this it will be done once a week.

Test Articles

Test Articles

Identification Suc/lac/man/sucralose (Cat. # 84097, L7877, #M9647, 7106Arespectively) Lot/Batch Number New sucrose has not arrived yet, 1085532,036935, 011B99, respectively Purity, Composition, Maintained bymanufacturer and Expiry Storage Conditions Room temperature Source andManufacturer Sigma Chemical Corporation, St. Louis, MO Special HandlingAppropriate PPE required (Lab coat, safety Precautions glasses, gloves).Prepared Immediately prior to dosing

Identification AT1001 (neat chemical, Groups 3, and 4) Lot/Batch NumberAT1001; Lot E050082 Purity and >95%; neat AT1001 Composition StorageConditions Frozen Source and Solvay/Peptisyntha Inc Manufacturer SpecialHandling Appropriate PPE required (Lab coat, Precautions safety glasses,and gloves) Prepared Freshly prepared each day prior to dosing

Identification Probiotic conditioned media Lot/Batch Number 5160D5Storage conditions Room temperature Source and manufacturer Sigma-TauPharmaceuticals Special Handling Use aseptic technique and appropriatePPE Precautions required (Lab coat, safety glasses, gloves). PreparedDissolve 0.1 g in 10 ml of MRS media, incubate at 37 C. for 24 hours.Filter through 0.22 μm membrane and dilute filtrate 1/10 in MRS media.Gavage animals with 30 μl.

Preparation of Test Articles

Suc/lac/man/suralose AT1001 AT1001 solution (neat chemical) (neatchemical) Test Article (Groups 1, 2, 3, and 4) (Group 3) (Group 4) Typeof Solution in water Oral solution in Oral solution in Formulationdrinking water drinking water Method of Weigh 50 g of sucrose, 6 g Weigh10 mg of neat Weigh 100 mg of Preparation of lactulose, 4 g of AT1001.Dissolve in neat AT1001. mannitol and 3 g of 100 mL sterile water.Dissolve in 100 mL sucralose and dissolve in sterile water. 100 ml ofsterile water. Frequency of Every week Every day Every day PreparationDose Sucrose 500 mg/ml 0.1 mg/ml 1 mg/ml Concentration Lactulose 60mg/ml Mannitol 40 mg/ml Sucralose 30 mg/ml Dose Volume 0.2 ml N/A N/AStorage Refrigerated Refrigerated Refrigerated Conditions

Test System

Species/Strain or Breed 129/Svev/IL-10 KO mouse Age at Study InitiationApproximately 28 days Weight Approximately 10 grams at study initiationand 20 grams at study termination Acclimation At least 3 days SelectionCriteria/ Randomized assignment of group and Randomization animal numberprior to study start Identification Ear markings and cage cards AnimalUse Protocol Number 138

Animals to be used on this study will be selected on the basis ofacceptable findings from physical examination and body weights. Theanimals will then be assigned to treatment groups prior to dosing.

Environmental Conditions

Caging Individually housed cages and in metabolism cages during urinecollection Bedding Direct bedding - wood chips prior to study startTemperature Approximate range 72 +/− 4° F. Humidity Range 30% to 70%Lighting Approximate 12-hour light, 12-hour dark cycle. The lightingcycle may be interrupted for performance of protocol-defined activities.Water Sterile filtered (0.22 micron filter) water Diet Certified PurinaRodent Meal 5001.

Clinical And Physical Examinations

Survival and Moribundity Throughout treatment periods of studyObservations Clinical Signs Once daily Unscheduled Observations To beperformed at the discretion of the study director/principal investigatorPhysical Examinations To be conducted once prior to the initiation ofdosing Routine Body Weights Prior to randomization and the first day ofevery week thereafter Food Consumption Two times a week. Mice will befasted 4 hours prior to each sugar gavage Water Consumption Every day.Stool Collection First day of every week and screened for the presenceof blood

Clinical Pathology

Urine Collection

During each intestinal permeability trial, all subjects will be gavagedwith suc/lac/man/sucralose solution and placed in metabolic cagesimmediately after to enable urine collection. Urine from each animalwill be collected for 22 hours in chilled collection tubes, treated with100 μL of a 10% Thymol solution (1.0 g/10 mL isopropanol) and paraffinoil (100 μL, to prevent urine evaporation) from the following intervalpost dosing 24 hours. Samples will be frozen at −80° C. until analysis.All sugars will be quantified by ion exchange HPLC. LAMA ratio, totalsucrose and total sucralose will be obtained per measurement.

Stool Sample Collection

On the first day of every week stool sample will be collected from everyanimal and screened for the presence of blood. Stool samples will alsobe collected at the termination of the study.

Sample Storage Conditions

50-100 μl serum from animals in Group 5 will be frozen until zonulinmeasurements made. 100 μl serum from animals from Groups 1-4 at Day 57and at day 77 will be frozen at −20° C. until zonulin measurements made.

Sacrifice Schedule

Animals found dead will be refrigerated and necropsied at the earliestpossible time (within working hours). Terminal body weight organ weightswill not be taken from animals found dead. Protocol defined tissues willbe collected.

Moribund/unscheduled animals that are sacrificed during normal workinghours will be taken immediately to necropsy. A terminal body weight willbe taken and the animal will be necropsied. Protocol defined tissues forhistology will be taken. Moribund/unscheduled sacrifice animals that aresacrificed outside of normal working hours will be refrigerated after aterminal body weight and necropsied at the earliest possible time(within working hours). Organ weights will not be collected unless theentire study is sacrificed early and control organ weights can becollected at the same time or the study is taken down at the scheduledsacrifice.

Animals Found Animals found dead will be refrigerated and Deadnecropsied at the earliest possible time (within working hours). Grossfindings will be noted. Moribund/ Moribund/unscheduled sacrifice animalsthat are Unscheduled sacrificed outside of normal working hours will beSacrifice refrigerated and necropsied at the earliest possible time(within working hours). Gross findings will be noted. Protocol-definedtissues will be collected. Sacrifice 1. At time 0, all 4 animals ofgroup 5 will be Schedule sacrificed. 50-100 μl serum will also becollected to measure zonulin levels. 2. After 8 weeks, on day 57, 4animals from the Groups 1-4 will be sacrificed and their intestinalpermeability (small bowel and colon) will be measured using Ussingchambers and samples from both sites will be assessed for histology, MPOand cytokine secretion. 100 μl of serum will be collected from theseanimals and sent to Alba Therapeutics for zonulin levels determination.3. On day 77, after final urine collection, all animals will besacrificed for measurements as above. Number of Animals All (survivalpermitting) Method of Cervical dislocation Euthanasia Fasting Animalswill be fasted overnight prior to necropsy Requirements Terminal BodyWill be taken at necropsy Weight Macroscopic Will be performed by thestudy pathologist at Examination necropsy

Immediately upon expiration, stomachs, small intestines, and colons willbe collected and weighed. Each tissue will be scored for macroscopiclesions to assess intestinal damage by the Study Pathologist. ELISAswill be performed on each tissue to measure the levels of MPO, IL-8,TNFα, and IFNγ. A section of the tissues will be fixed in formalin forHE histology. The day after the final intestinal permeability measureson day 77, all surviving animals will be euthanized and intestinaltissues collected and processed.

Clinical observations, physical examinations, and body weights will berecorded on appropriate paper forms. Sucrose, lactulose, mannitol, andsucralose and lactulose:mannitol ratios will be determined according tothe published procedures.

Data Acquisition

The following data will be acquired

Data Type Schedule Weights 1. Weigh all animals on the first day ofevery week 2. Weigh all animals at sacrifice 3. Week 8 Sacrifice 4animals from each group to dissect out stomachs, small intestines, andcolons and weigh them 4. Week 11 Sacrifice the remaining 8 animals fromeach group to dissect out stomachs, and intestines, and colons and weighthem Suc/lac/manLAMA 1. Weeks 1-2 in urine AdministerSuc/lac/man/suralose and collect urine for 22 hours on days 3, 6, 9 2.Weeks 3-11 Administer Suc/lac/man/suralose and collect urine urine for22 hours on days 14, 21, 28, 35, 41, 49, 56, 63, 70, 77 Intestinal 1.Week 8 - Day 57 of study permeability with Sacrifice 4 animals from eachgroup to dissect out stomachs, small Ussing chambers intestines andcolons and measure intestinal permeability 2. Week 11 - termination ofstudy at day 77 Sacrifice the remaining 8 animals from each group todissect out stomachs, small intestines and colons and measure intestinalpermeability Scoring lesions 1. Day 1 histologically Dissect outstomachs, small intestines and colons of 4 animals in Group 5 forscoring macroscopic lesions as control, fix sections for histology. 2.Week 8 - Day 57 of study Dissect out stomachs, small intestines andcolons of 4 animals from Groups 1-4 for scoring macroscopic lesions, fixsections for histology 3. Week 11- Day 77 of study Dissect out stomachs,small intestines and colons of remaining 8 animals from Groups 1-4 forscoring macroscopic lesions, fix sections for histology Zonulinlevels 1. Sacrifice all 4 animals from Group 5 to measure zonulin levelsday 1 2. Week 8 - Day 57 of study Measure zonulin levels in the animalsfrom Groups 1-4 sacrificed on this day 2. Week 11- Day 77 of studyMeasure zonulin levels in the animals from Groups 1-4 sacrificed on thisday Levels of MPO, 1. Measure levels of these proteins in animals fromGroup 5 IL-8, TNF-α, IFNγ 2. Measure levels of these proteins in animalsfrom Groups 1-4 at the beginning of Week 8 - Day 57 of study 3. Measurelevels of these proteins in animals from Groups 1-4 when study isterminated at Week 11- Day 77 of study Water consumption 1. Measurewater consumption daily for dose measurement

FIGS. 1-4 show that development of disease is associated with anincrease in small intestinal permeability. This increase can beabrogated by high dose AT-1001.

FIG. 5-10 show the results of analysis of disease in the colon. Diseasein the colon was evaluated at both 8 and 17 week time points. The formerwith Using chamber measurements and the latter with histology, mucosalcytokine secretion, MPO and sucralose permeability. At 8 weeks of ageAT-1001 reduced colonic permeability to mannitol and prevented thereduction in electrical resistance observed in the untreated animals. At17 weeks AT-1001 reduced all tissue markers of colonic inflammation thatwere measured

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated by reference.

1. A method for treating Inflammatory Bowel Disease (IBD) in a patientin need thereof, the method comprising: administering a compositioncomprising a peptide having the amino acid sequence GGVLVQPG (SEQ IDNO:15) to the duodenum of the patient, wherein the peptide is beingadministered in a delayed release composition that is stable in gastricfluid and unstable in intestinal fluid so as to release an effectiveamount of the peptide in the duodenum of the patient.
 2. The method ofclaim 1, wherein the patient has Crohn's Disease.
 3. The method of claim1, wherein the peptide is further administered to the jejunum of thepatient.
 4. The method of claim 1, wherein at least 70% by weight of thepeptide is released by the composition in from 5 to 90 minutes ofexposure to simulated intestinal fluid having a pH of greater than
 5. 5.The method of claim 1, wherein at least 70% by weight of the peptide isreleased by the composition by about 60 minutes of exposure to simulatedintestinal fluid having a pH of greater than
 5. 6. The method of claim1, further comprising, administering an active agent selected from anaminosalicylate, corticosteroid, immunomodulator, or antibiotic.
 7. Themethod of claim 6, wherein the aminosalicylate is 5-aminosalicyclic acid(5-ASA).
 8. The method of claim 1, wherein the peptide is administeredone or more times per day.
 9. The method of claim 8, wherein the peptideis administered chronically.
 10. The method of claim 1, wherein thesubject is not undergoing an acute Inflammatory Bowel Disease (IBD)attack at the time the peptide is administered.
 11. The method of claim1, wherein the subject is undergoing an acute attack of InflammatoryBowel Disease (IBD) at the time the peptide is administered.
 12. Themethod of claim 1, wherein the patient is human.
 13. The method of claim1, wherein the patient is a dog.
 14. A method for treating chronicInflammatory Bowel Disease (IBD) in a patient in need thereof, themethod comprising: administering a composition comprising a peptidehaving the amino acid sequence GGVLVQPG (SEQ ID NO: 15) in an effectiveamount to the patient one or more times per day for a plurality of days,wherein the peptide is being administered in a delayed releasecomposition that is stable in gastric fluid and unstable in intestinalfluid so as to release the peptide in the duodenum and jejunum of thepatient.